The transfer of genes into cells provides a means to determine gene function and treat diseases having a genetic basis. In addition, gene transfer provides the basis for high-level protein expression, used by molecular researchers to study protein function and to produce new protein drugs. The introduction of genes into animals can also produce useful animal models of human diseases.
Many methods have been developed to introduce exogenous genes into cells. The earliest method for introducing DNA into cells was to incubate the DNA with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE) had been coupled. These large DNA-containing particles stuck to cells and were taken up by endocytosis. However, this method was inefficient for the introduction of nucleic acid into many types of cells. Another widely used method is the precipitation of DNA with calcium phosphate, which allows transient expression of genes in cells. However, neither of these methods allow the transfer of nucleic acids into a cell with a high efficiency, nor do they allow for persistent gene expression. Thus, a need remains for a high efficiency method for the stable introduction of genes into cells.
Viral vectors, such as adenoviral or retroviral vectors, have been used to introduce foreign DNA with high efficiency. Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. After a retrovirus infects a host cell, the retroviral genomic RNA is reverse transcribed to the double stranded DNA form. This DNA can integrate into the host genome to form a provirus. Reverse transcription requires cis-acting viral sequences including the primer binding site (pbs), the repeat (R) region of the long terminal repeats (LTRs), and the polypurine tract (ppt). Viral terminal attachment sites (att) mediate the integration of the provirus into the host genome. The integrated provirus is transcribed into full-length and spliced mRNA These RNAs are used as templates to translate viral proteins. Full length mRNA is packaged by the viral proteins, which recognize viral RNA by the cis-acting E or ψ sequence. The viral particles, or virions, exit the cell by budding from the cell membrane.
Retroviral vectors containing parts of the retrovirus are used to introduce foreign DNA into eukaryotic cells. Retroviral vectors usually contain the cis-acting sequences required for packaging, reverse transcription, and integration. However, these vectors are replication incompetent because they are defective in retroviral structural genes. Helper cells containing helper virus DNA supply the deficient viral gene products. Thus by transfecting the replication incompetent retrovirus into helper cells, retroviral RNA can be packaged and released as vector virus particles. Because the helper virus is deficient in cis-acting functions, its RNA is not packaged, and helper-free viral stocks can be produced. The released vector-containing particles can be used to introduce the foreign DNA into target cells.